Quantitative trait loci (QTLs) are chromosomal regions containing genes that influence a complex trait such as alcohol (ethanol) withdrawal severity. We have mapped several QTLs that jointly have a major influence on the severity of alcohol withdrawal in populations derived from the C57BL/6Jo(B6) and DBA/2J (D2) inbred mouse strains. The three largest QTLs (all LOD > 5, p < 2x10 -6) are on distal chromosome 1, mid chromosome 4, and proximal chromosome 11. Based on mouse-human homology, the human counterparts of the three mouse QTLs map to human chromosomes lq22-32, 9p24-23, and 5q31-35, respectively. During the current period of AAl1114, we have identified Kcnj9, Mpdz, and Gabrg2 as promising candidate genes for the mouse chromosome 1, 4 and 11 QTLs, respectively. These promising candidate genes map within the QTL interval and show genotype-dependent differences in gene expression and/or coding sequence (structural differences) that may underlie the QTL. Kcnj9 encodes GIRK3, a member of the G-protein-activated inwardly rectifying potassium channel family. Mpdz encodes a multiple PDZ domain protein. Gabrg2 encodes the GABAA receptor gamma 2 subunit. For the gene(s) underlying a QTL, genotype-dependent (allelic) differences in gene expression and/or coding sequence must be functionally relevant at the protein level, e.g., affect protein expression and/or functional activity. The proposed work will use innovative genetic animal models to test the hypothesis that genotype-dependent differences in these promising candidate genes are functionally relevant at the protein level and influence alcohol withdrawal severity. Using congenic strains to isolate each of the three QTLs against a uniform (inbred) genetic background, and transgenic animal models, we propose to continue toward identification and eventual proof of the genes that underlie each QTL. To accomplish this, we propose the following. (1) Promising candidate genes will be tested for differences between the congenic strain and background strain in their protein product expression using Western blot analysis. (2) Promising candidate genes will be tested for differences between the congenic strain and background strain in the functional activity of their protein products. (3) Kcnj9 (GIRK3) knockout and their wild-type littermates will be behaviorally tested for alcohol withdrawal severity. (4) Conventional and conditional Gabrg2 knockdown mice (i.e., heterozygotes derived from gamma 2 subunit knockout mice) and their wild-type littermates will be behaviorally tested for alcohol withdrawal severity